Remarkable progress has been made in the past few years in understanding the molecular mechanisms of damage-induced mutagenesis. It has been suggested that a significant proportion of mutations arise when damaged genomic DNA is replicated in an error-prone manner by one or more low-fidelity polymerases (Goodman et al., Annu. Rev. Biochem. 71:17-50, 2002). These polymerases appear to have evolved to specifically facilitate replication of a wide variety of DNA lesions that might otherwise block the high fidelity replication machinery. Most of these specialized polymerases are phylogenetically related to each other and have been collectively termed “Y-family” polymerases (Ohmori et al., Mol. Cell. 8:7-8, 2001).
The Y-family polymerases are ubiquitous and are found in all three kingdoms of life, with many organisms often possessing more than one family member. This suggests that Y-family polymerases play important roles in cellular survival or evolutionary “fitness” (Friedberg et al., Science 296:1627-30, 2002; Yeiser et al., Proc. Natl. Acad. Sci. USA 99:8737-41, 2002). Indeed, defects in human Polη result in the sunlight-sensitive and cancer prone xeroderma pigmentosum variant (XP-V) syndrome (Masutani et al., Nature 399:700-04, 1999; Johnson et al., Science 285:263-65, 1999), whilst mutations in Escherichia coli dinB reduces the cell's ability to undergo adaptive mutagenesis in stationary phase (McKenzie et al., Mol. Cell. 7:571-79, 2001; Tompkins et al., J. Bacteriol. 185:3469-72, 2003).
In vivo, DNA polymerases participate in a spectrum of DNA synthetic processes including DNA replication, DNA repair, recombination, and gene amplification (Kornberg and Baker, DNA Replication, pp. 929, W.H. Freeman and Co., New York, 1992). In vitro, DNA polymerases are used for DNA amplification techniques, for example polymerase chain reaction (PCR). DNA polymerases, particularly thermostable polymerases, are the key to a large number of techniques in recombinant DNA studies and in medical diagnosis of disease.
Due to the importance of Y-family polymerases in biotechnology and medicine, it would be advantageous to identify other thermostable Y-family polymerases and to create chimeric Y-family polymerases in order to optimize polymerase characteristics such as thermostability, fidelity, processivity, and translesion synthesis.